Project Summary/Abstract: There are more than 11 million people in the world with keloids and more than 425,000 associated clinic visits, yearly, in the United States. Keloids are benign fibroproliferative tumors which cause pain, pruritus, emotional distress and loss of function. Current therapies are unsatisfactory with unacceptably high recurrence rates, mainly because of an incomplete understanding of keloid pathogenesis. Fibroblasts are a key player in keloid pathogenesis, but the drivers are unknown. Keloid disease is influenced by aberrant signaling pathways. However, no clear signaling pathway has been identified. Exosomes mediate cell-cell communication, exercising primary physiological and pathophysiological function. Exosomal cargo, such as microRNAs (miRNAs), regulate cellular function. Our group identified RAB27, important for exosome secretion, as being differentially hypomethylated in keloid compared to normal skin. Our group has isolated keloid-specific exosomes. To date, there are no published studies on keloid exosomes or the contribution of RAB27 methylation on exosome function. We propose to test the central hypothesis that exosomes communicate critical signaling events in the keloid microenvironment mediated by RAB27 gene methylation. Aim 1: To determine the effect of RAB27 methylation on keloid exosome production and miRNA cargo profiles. Hypotheses: (1) Keloid exosomal production correlates with RAB27 gene methylation. (2) Keloid exosomal cargo miRNA expression profiles correlates with RAB27 gene methylation. (3) Keloid exosome miRNA?s putative target genes lie within pathways essential for wound healing and/or fibrosis. Aim 2: To determine the effects of keloid exosomes on the keloid microenvironment. Hypothesis: Keloid fibroblast exosomes compared to normal fibroblast exosomes will cause pro-fibrotic phenotype changes in normal fibroblasts. Aim 3: To determine the effect of keloid exosomes on scar formation in vivo. Hypothesis: Exosomes generated in aim 1 and tested in aim 2 will increase scar formation in a rabbit ear scar model. Significance: This project will lead to an enhanced understanding of keloid pathogenesis and the potential for exosome-based therapy. Innovation: (i) rational progression from preliminary data supporting the novel role of exosomes in keloid pathogenesis; (ii) investigating the influence of RAB27 methylation on the function and production of keloid exosomes would suggest a mechanistic basis for novel epigenetic biomarkers;(iii) using unique resources which includes fibroblast cell lines from primary untreated keloid (25) and matched normal skin (25) from a multi-ethnic group of patients and an in vivo animal model allow for the pragmatic translational application of results; (iv) entirely new field of keloid investigation. In summary, this project, mentoring and career development plan will position, Lamont R Jones, MD, MBA, to become an independent clinician scientist and leader in keloid pathogenesis.